Nguyen Hoang Loc

Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1–ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

Modification of the cholera toxin B subunit coding sequence to enhance expression in plants

The cholera toxin B subunit (CTB) contains five identical polypeptides and targets glycosphingolipid receptors on eukaryotic cell surfaces. Increased expression of CTB in plants is critical for the development of edible vaccines. In this study, the coding sequence of the CTB gene was optimized, based on the modification of codon usage to that of tobacco plant genes and the removal of mRNA-destabilizing sequences. The synthetic CTB gene was cloned into a plant expression vector and expressed in tobacco plants under the control of the CaMV 35S promoter. The recombinant CTB protein constituted approximately 1.5% of the total soluble protein in transgenic tobacco leaves. This level of CTB production was approximately 15-fold higher than that in tobacco plants that were transformed with the bacterial CTB gene. The recombinant CTB produced by tobacco plants demonstrated strong affinity for GM1-ganglioside, which indicates that the sites required for binding and proper folding of the pentameric CTB structure were conserved. This is the first report on the optimization of the CTB-coding sequence to give a dramatic increase in CTB expression in plants.

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andKhGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andKhGM-CSF of 7.64 after 2 h of incubation at room temperature.

Expression of the Escherichia coli heat-labile enterotoxin B subunit in transgenic watercress (Nasturtium officinale L.)

A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. GM1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.

Tissue culture and expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic Peperomia pellucida.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a non-toxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. We synthesized a gene encoding the LTB adapted to the optimized coding sequences in plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its expression level and protein assembly in plants. The synthetic LTB gene was located into a plant expression vector under the control of CaMV 35S promoter and was introduced into Peperomia pellucida by biolistic transformation method. The integration of synthetic LTB gene into genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification method. The assembly of plant-produced LTB was detected by western blot analysis. The amount of LTB protein produced in transgenic P. pellucida leaves was approximately 0.75% of the total soluble plant protein. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is receptor for LTB on the cell surface, suggesting that the LTB subunits formed biological active pentamers.

Production of asiaticoside from centella (Centella asiatica L. Urban) cells in bioreactor

OBJECTIVE To investigate the effects of some culture conditions on production of asiaticoside from centella (Centella asiatica L. Urban) cells cultured in 5-L bioreactor. METHODS The centell cell suspension culture was conducted in 5-L bioreactor to investigate the growth and asiaticoside accumulation under various conditions. Asiaticoside content was determined by HPLC analysis. RESULTS The results showed that the cell growth and asiaticoside accumulation peaked after 24 d of culture at an agitation speed of 150 r/min and aeration rate of 2.5 L/min. The cell biomass reached a maximum value of 302.45 g fresh weight (31.45 g dry weight) and growth index of 3.03 with inoculum size of 100 g. However, asiaticoside content was the highest (60.08 mg/g dry weight) when culture was initiated with an inoculum size of 50 g. CONCLUSIONS The present study found the suitable conditions for growth of centella cells and their asiaticoside production in bioreactor.

Trichoderma asperellum Chi42 Genes Encode Chitinase

Four Trichoderma strains (CH2, SH16, PQ34, and TN42) were isolated from soil samples collected from Quang Tri and Thua Thien Hue provinces in Vietnam. The strains exhibited high chitinolytic secretion. Strain PQ34 formed the largest zone of chitinase-mediated clearance (> 4 cm in diameter) in agar containing 1% (w/v) colloidal chitin. Analysis of the internal transcribed spacer regions of these strains indicated that they were Trichoderma asperellum. The molecular weights of the chitinases were approximately 42 kDa. Chitinase genes (chi42) of T. asperellum strains TN42, CH2, SH16, and PQ34 were 98~99% homologous to the ech42 gene of T. harzianum CB-Pin-01 (accession No. DQ166036). The deduced amino acid sequences of both T. asperellum strains SH16 and TN42 shared 100% similarity.

Effects of elicitors on the enhancement of asiaticoside biosynthesis in cell cultures of centella ( Centella asiatica L. Urban)

In this work, the effects of elicitor concentration and elicitation day on the growth and asiaticoside production of centella cells were investigated. The results showed that 2-hydroxybenzoic acid from 50–200 μM and yeast extract from 2–5 g L−1 had different eliciting influences. The addition of 2-hydroxybenzoic acid and yeast extract to the cultures strongly enhanced asiaticoside production in centella cells. The increase in asiaticoside content induced by the addition of 100 μM of 2-hydroxybenzoic acid and 4 g L−1 of yeast extract at day 10 of inoculation was about 5- and 3.5-fold, respectively, as compared with that of the reference cells. In general, 2-hydroxybenzoic acid (abiotic elicitor) was more effective in enhancing asiaticoside biosynthesis than yeast extract (biotic elicitor).

Cloning and expression of two genes coding endo-β-1,4-glucanases from Trichoderma asperellum PQ34 in Pichia pastoris

Two genes coding endo-β-1,4-glucanases were cloned from Trichoderma asperellum PQ34 which was isolated from Thua Thien Hue province, Vietnam. The expression of these genes in Pichia pastoris produced two enzymes with molecular masses of approximately 46 kDa (about 42 kDa of enzymes and 4 kDa of signal peptide). The effects of induction time and temperature, inducer concentration, and culture medium on the endo-β-1,4-glucanase activity were investigated. The results showed that the highest total activities of two endo-β-1,4-glucanases were approximately 4.7 × 10−8 kat (from Glu1-TA gene) and 7.3 × 10−8 kat (from Glu2-TA gene) occurred after 4 days of induction using 25 mL L−1 methanol at 30◦C when the yeast cells were cultured in a YPL medium.

Enhancing expression of recombinant fimbrial adhesin K88 isolated of enterotoxigenic Escherichia coli from piglet

Fimbrial adhesins are important toxic factors of enterotoxigenic E. coli (ETEC) strains, whereby they have the ability to adhere to small intestine of the host. K88 (F4) is the first fimbrial adhesin was detected in ETEC strains from piglet, and it is the most popular fimbrial adhesin of E. coli strains causes diarrhea in weaned piglets. In previous report, we successful expressed the fae G gene encoding fimbrial adhesin K88-1NT in recombinant E. coli BL21 (DE3). Highest level of K88-1NT was determined after induction by 0.1% lactose for 6 h at 35 o C when cell density (OD 600 ) reached a value of 0.5. Induction medium including Na 2 HPO 4 .12H 2 O 1%, KH 2 PO 4 0.3%, NaCl 0.05%, (NH 4 ) 2 SO 4 1%, MgSO 4 .7H 2 O 0.2%, glucose 1.5%, tryptone 1% and yeast extract 1%. Purified K88-1NT protein from Ni 2+ affinity chromatography had a molecular weight of 31 kDa.